Apolipoprotein M increases the expression of vitamin D receptor mRNA in colorectal cancer cells detected with duplex fluorescence reverse transcription-quantitative polymerase chain reaction

Yu,Miao‑Mei; Yao,Shuang; Luo,Kai‑Ming; Mu,Qin‑Feng; Yu,Yang; Luo,Guang‑Hua; Xu,Ning

doi:10.3892/mmr.2017.6716

Apolipoprotein M increases the expression of vitamin D receptor mRNA in colorectal cancer cells detected with duplex fluorescence reverse transcription-quantitative polymerase chain reaction

Authors:
- Miao‑Mei Yu
- Shuang Yao
- Kai‑Ming Luo
- Qin‑Feng Mu
- Yang Yu
- Guang‑Hua Luo
- Ning Xu
View Affiliations

Affiliations: Comprehensive Laboratory, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu 213003, P.R. China, Section of Clinical Chemistry and Pharmacology, Institute of Laboratory Medicine, Lunds University, S‑22185 Lund, Sweden
Published online on: June 7, 2017 https://doi.org/10.3892/mmr.2017.6716
Pages: 1167-1172
Copyright: © Yu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Apolipoprotein M (ApoM) and the vitamin D receptor (VDR) are apolipoproteins predominantly presenting in high-density lipoprotein (HDL) and a karyophilic protein belonging to the steroid‑thyroid receptor superfamily, respectively. Previous studies have demonstrated that ApoM and VDR are associated with cholesterol metabolism, immune and colorectal cancer regulation. In order to investigate whether ApoM affected the expression of VDR in colorectal cancer cells, a single‑tube duplex fluorescence reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) system was developed to simultaneously detect the mRNA levels of VDR and GAPDH in HT‑29 cells overexpressing ApoM. The results demonstrated that the amplification products were confirmed as the specific fragment of VDR/GAPDH using the DNA sequencing instrument. The sensitivity, linear range, correlation coefficient, amplification efficiency, intra‑assay and inter‑assay coefficients of variation were 40 copies/µl, 4.00x101‑4.00x105 copies/µl, 0.999, 92.42%, 0.09‑0.34% and 0.32‑0.65% for VDR, and 40 copies/µl, 4.00x101‑4.00x105 copies/µl, 0.999, 98.07%, 0.19‑0.43% and 0.40‑0.75% for GAPDH, respectively. The results indicated that the expression of VDR mRNA was significantly higher in HT‑29 cells overexpressing ApoM, compared with the negative control group (P<0.05). In conclusion, the current study successfully developed the single‑tube duplex RT‑qPCR to simultaneously detect VDR and GAPDH expression in colorectal cancer cells. The methodology results demonstrated that the duplex RT‑qPCR system with high sensitivity and specificity could ensure the objectivity and credibility of the detection. The present study confirmed that ApoM significantly increased the expression of VDR in HT‑29 cells. In addition, it was hypothesized that ApoM may be involved in antineoplastic activity via the upregulation of VDR expression, which may provide novel directions for the investigation of ApoM in cancer.

View Figures

View References

1	Xu N and Dahlbäck B: A novel human apolipoprotein (apoM). J Biol Chem. 274:31286–31290. 1999. View Article : Google Scholar : PubMed/NCBI
2	Wolfrum C, Poy MN and Stoffel M: Apolipoprotein M is required for prebeta-HDL formation and cholesterol efflux to HDL and protects against atherosclerosis. Nat Med. 11:418–422. 2005. View Article : Google Scholar : PubMed/NCBI
3	Huang XS, Zhao SP, Hu M and Luo YP: Apolipoprotein M likely extends its anti-atherogenesis via anti-inflammation. Med Hypotheses. 69:136–140. 2007. View Article : Google Scholar : PubMed/NCBI
4	Arkensteijn BW, Berbée JF, Rensen PC, Nielsen LB and Christoffersen C: The apolipoprotein m-sphingosine-1-phosphate axis: Biological relevance in lipoprotein metabolism, lipid disorders and atherosclerosis. Int J Mol Sci. 14:4419–4431. 2013. View Article : Google Scholar : PubMed/NCBI
5	Luo G, Zhang X, Mu Q, Chen L, Zheng L, Wei J, Berggren-Söderlund M, Nilsson-Ehle P and Xu N: Expression and localization of apolipoprotein M in human colorectal tissues. Lipids Health Dis. 9:1022010. View Article : Google Scholar : PubMed/NCBI
6	Gonzalez-Parra E, Rojas-Rivera J, Tuñón J, Praga M, Ortiz A and Egido J: Vitamin D receptor activation and cardiovascular disease. Nephrol Dial Transplant. 27 Suppl 4:iv17–21. 2012. View Article : Google Scholar : PubMed/NCBI
7	Christakos S, Dhawan P, Verstuyf A, Verlinden L and Carmeliet G: Vitamin D: Metabolism, molecular mechanism of action, and pleiotropic effects. Physiol Rev. 96:365–408. 2016. View Article : Google Scholar : PubMed/NCBI
8	Li S, He Y, Lin S, Hao L, Ye Y, Lv L, Sun Z, Fan H, Shi Z, Li J, et al: Increase of circulating cholesterol in vitamin D deficiency is linked to reduced vitamin D receptor activity via the Insig-2/SREBP-2 pathway. Mol Nutr Food Res. 60:798–809. 2016. View Article : Google Scholar : PubMed/NCBI
9	Dou R, Ng K, Giovannucci EL, Manson JE, Qian ZR and Ogino S: Vitamin D and colorectal cancer: Molecular, epidemiological and clinical evidence. Br J Nutr. 115:1643–1660. 2016. View Article : Google Scholar : PubMed/NCBI
10	Feldman D, Krishnan AV, Swami S, Giovannucci E and Feldman BJ: The role of vitamin D in reducing cancer risk and progression. Nat Rev Cancer. 14:342–357. 2014. View Article : Google Scholar : PubMed/NCBI
11	Nurmi J, Ylikoski A, Soukka T, Karp M and Lövgren T: A new label technology for the detection of specific polymerase chain reaction products in a closed tube. Nucleic Acids Res. 28:E282000. View Article : Google Scholar : PubMed/NCBI
12	Angelone-Alasaad S, Min A Molinar, Pasquetti M, Alagaili AN, D'Amelio S, Berrilli F, Obanda V, Gebely MA, Soriguer RC and Rossi L: Universal conventional and real-time PCR diagnosis tools for Sarcoptes scabiei. Parasit Vectors. 8:5872015. View Article : Google Scholar : PubMed/NCBI
13	Parker J, Fowler N, Walmsley ML, Schmidt T, Scharrer J, Kowaleski J, Grimes T, Hoyos S and Chen J: Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses. PLoS One. 10:e01431642015. View Article : Google Scholar : PubMed/NCBI
14	Shen Z: Effect of reaction tube on the accuracy of polymerase chain reaction. J Prac Medi Tech. 17:142–143. 2010.
15	Gubelmann C, Gattiker A, Massouras A, Hens K, David F, Decouttere F, Rougemont J and Deplancke B: GETPrime: A gene- or transcript-specific primer database for quantitative real-time PCR. Database (Oxford). 2011:bar0402011. View Article : Google Scholar : PubMed/NCBI
16	Wang X, Spandidos A, Wang H and Seed B: PrimerBank: A PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Res. 40(Database issue): D1144–D1149. 2012. View Article : Google Scholar : PubMed/NCBI
17	Huang LZ, Gao JL, Pu C, Zhang PH, Wang LZ, Feng G and Zhang Y: Apolipoprotein M: Research progress, regulation and metabolic functions (Review). Mol Med Rep. 12:1617–1624. 2015.PubMed/NCBI
18	Elsøe S, Christoffersen C, Luchoomun J, Turner S and Nielsen LB: Apolipoprotein M promotes mobilization of cellular cholesterol in vivo. Biochim Biophys Acta. 1831:1287–1292. 2013. View Article : Google Scholar : PubMed/NCBI
19	Luo G, Zhang X, Nilsson-Ehle P and Xu N: Apolipoprotein M. Lipids Health Dis. 3:212004. View Article : Google Scholar : PubMed/NCBI
20	Matusiak D, Murillo G, Carroll RE, Mehta RG and Benya RV: Expression of vitamin D receptor and 25-hydroxyvitamin D3-1{alpha}-hydroxylase in normal and malignant human colon. Cancer Epidemiol Biomarkers Prev. 14:2370–2376. 2005. View Article : Google Scholar : PubMed/NCBI
21	Milczarek M, Filip-Psurska B, Swiętnicki W, Kutner A and Wietrzyk J: Vitamin D analogs combined with 5-fluorouracil in human HT-29 colon cancer treatment. Oncol Rep. 32:491–504. 2014.PubMed/NCBI
22	Elimrani I, Koenekoop J, Dionne S, Marcil V, Delvin E, Levy E and Seidman EG: Vitamin D Reduces Colitis- and Inflammation-Associated Colorectal Cancer in Mice Independent of NOD2. Nutr Cancer. 69:276–288. 2017. View Article : Google Scholar : PubMed/NCBI
23	Vidigal VM, Silva TD, de Oliveira J, Pimenta CAM, Felipe AV and Forones NM: Genetic polymorphisms of vitamin D receptor (VDR), CYP27B1 and CYP24A1 genes and the risk of colorectal cancer. Int J Biol Markers. 32:e224–e230. 2017. View Article : Google Scholar : PubMed/NCBI
24	Takada I and Makishima M: Control of inflammatory bowel disease and colorectal cancer by synthetic vitamin D receptor ligands. Curr Med Chem. Dec 2–2016.(Epub ahead of print).
25	Yazdani S, Poosti F, Toro L, Wedel J, Mencke R, Mirković K, de Borst MH, Alexander JS, Navis G, van Goor H, et al: Vitamin D inhibits lymphangiogenesis through VDR-dependent mechanisms. Sci Rep. 7:444032017. View Article : Google Scholar : PubMed/NCBI
26	Chiang KC, Yeh TS, Huang CC, Chang YC, Juang HH, Cheng CT, Pang JS, Hsu JT, Takano M, Chen TC, et al: MART-10 represses cholangiocarcinoma cell growth and high vitamin D receptor expression indicates better prognosis for cholangiocarcinoma. Sci Rep. 7:437732017. View Article : Google Scholar : PubMed/NCBI
27	Tavera-Mendoza LE, Westerling T, Libby E, Marusyk A, Cato L, Cassani R, Cameron LA, Ficarro SB, Marto JA, Klawitter J and Brown M: Vitamin D receptor regulates autophagy in the normal mammary gland and in luminal breast cancer cells. Proc Natl Acad Sci USA. 114:E2186–E2194. 2017. View Article : Google Scholar : PubMed/NCBI